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Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers

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ABSTRACT

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

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Increased sensitivity of melanoma cells to CPEB4 depletion.(a) Immunoblots showing CPEB4 levels upon transduction of control or CPEB4 shRNAs in a panel of melanoma (red) and non-melanoma (black) cell lines. (b) Colony formation assays of the indicated tumour cell lines seeded at high or low density (5 × 104 or 2–4 × 103 cells, respectively). Bar graphs correspond to cell number estimated by crystal violet staining and represented with respect to shC-transfected cells. Data are plotted as means±s.e.m. of three independent experiments in duplicate. (c) Depletion of CPEB4 (by shRNA) in genetically matched pairs of human skin fibroblasts and melanocytes visualized by immunoblotting (upper) with respect to shC-transduced cells. The human melanoma cell line UACC-62 is included as a reference. Data are represented as means±s.e.m. of two experiments in triplicate. (d) Micrographs showing morphological changes driven by shCPEB4 in primary fibroblasts, melanocytes and UACC-62 melanoma cells. Scale bars, 20 μm (unless otherwise indicated). ***P<0.001, *P<0.05. NS, non-significant.
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f3: Increased sensitivity of melanoma cells to CPEB4 depletion.(a) Immunoblots showing CPEB4 levels upon transduction of control or CPEB4 shRNAs in a panel of melanoma (red) and non-melanoma (black) cell lines. (b) Colony formation assays of the indicated tumour cell lines seeded at high or low density (5 × 104 or 2–4 × 103 cells, respectively). Bar graphs correspond to cell number estimated by crystal violet staining and represented with respect to shC-transfected cells. Data are plotted as means±s.e.m. of three independent experiments in duplicate. (c) Depletion of CPEB4 (by shRNA) in genetically matched pairs of human skin fibroblasts and melanocytes visualized by immunoblotting (upper) with respect to shC-transduced cells. The human melanoma cell line UACC-62 is included as a reference. Data are represented as means±s.e.m. of two experiments in triplicate. (d) Micrographs showing morphological changes driven by shCPEB4 in primary fibroblasts, melanocytes and UACC-62 melanoma cells. Scale bars, 20 μm (unless otherwise indicated). ***P<0.001, *P<0.05. NS, non-significant.

Mentions: The results above suggested that in contrast to the non-cell autonomous roles of CPEB4 in pancreatic cancer (related to remodelling of the stroma)35, this protein could exert a more pressing role in cell proliferation. Therefore, the depletion of CPEB4 was assessed in melanoma cells cultured as monolayers in the absence of other cell types (Fig. 2c). In all cases tested the two CPEB4 shRNAs promoted an acute abrogation of cell proliferation (Fig. 2c), with characteristic features of premature senescence, as determined by staining for β-galactosidase activity at acidic pH (Fig. 2d,e). These effects were in contrast to the minor impact of CPEB4 downregulation in the pancreatic cancer RWP1 (Fig. 3a,b). Similarly, melanoma cells were significantly more dependent on CPEB4 than HeLa, U251 and 639 V (Fig. 3a,b), selected as examples of cell lines from cervical carcinoma, glioblastoma and epithelial sites (bladder cancer), where CPEB4 roles have been best described41735.


Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers
Increased sensitivity of melanoma cells to CPEB4 depletion.(a) Immunoblots showing CPEB4 levels upon transduction of control or CPEB4 shRNAs in a panel of melanoma (red) and non-melanoma (black) cell lines. (b) Colony formation assays of the indicated tumour cell lines seeded at high or low density (5 × 104 or 2–4 × 103 cells, respectively). Bar graphs correspond to cell number estimated by crystal violet staining and represented with respect to shC-transfected cells. Data are plotted as means±s.e.m. of three independent experiments in duplicate. (c) Depletion of CPEB4 (by shRNA) in genetically matched pairs of human skin fibroblasts and melanocytes visualized by immunoblotting (upper) with respect to shC-transduced cells. The human melanoma cell line UACC-62 is included as a reference. Data are represented as means±s.e.m. of two experiments in triplicate. (d) Micrographs showing morphological changes driven by shCPEB4 in primary fibroblasts, melanocytes and UACC-62 melanoma cells. Scale bars, 20 μm (unless otherwise indicated). ***P<0.001, *P<0.05. NS, non-significant.
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f3: Increased sensitivity of melanoma cells to CPEB4 depletion.(a) Immunoblots showing CPEB4 levels upon transduction of control or CPEB4 shRNAs in a panel of melanoma (red) and non-melanoma (black) cell lines. (b) Colony formation assays of the indicated tumour cell lines seeded at high or low density (5 × 104 or 2–4 × 103 cells, respectively). Bar graphs correspond to cell number estimated by crystal violet staining and represented with respect to shC-transfected cells. Data are plotted as means±s.e.m. of three independent experiments in duplicate. (c) Depletion of CPEB4 (by shRNA) in genetically matched pairs of human skin fibroblasts and melanocytes visualized by immunoblotting (upper) with respect to shC-transduced cells. The human melanoma cell line UACC-62 is included as a reference. Data are represented as means±s.e.m. of two experiments in triplicate. (d) Micrographs showing morphological changes driven by shCPEB4 in primary fibroblasts, melanocytes and UACC-62 melanoma cells. Scale bars, 20 μm (unless otherwise indicated). ***P<0.001, *P<0.05. NS, non-significant.
Mentions: The results above suggested that in contrast to the non-cell autonomous roles of CPEB4 in pancreatic cancer (related to remodelling of the stroma)35, this protein could exert a more pressing role in cell proliferation. Therefore, the depletion of CPEB4 was assessed in melanoma cells cultured as monolayers in the absence of other cell types (Fig. 2c). In all cases tested the two CPEB4 shRNAs promoted an acute abrogation of cell proliferation (Fig. 2c), with characteristic features of premature senescence, as determined by staining for β-galactosidase activity at acidic pH (Fig. 2d,e). These effects were in contrast to the minor impact of CPEB4 downregulation in the pancreatic cancer RWP1 (Fig. 3a,b). Similarly, melanoma cells were significantly more dependent on CPEB4 than HeLa, U251 and 639 V (Fig. 3a,b), selected as examples of cell lines from cervical carcinoma, glioblastoma and epithelial sites (bladder cancer), where CPEB4 roles have been best described41735.

View Article: PubMed Central - PubMed

ABSTRACT

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

No MeSH data available.


Related in: MedlinePlus